PROJECT SUMMARY Urinary tract infection (UTI) is a costly clinical problem that affects millions of women worldwide. Clinical studies implicate bacterial vaginosis (BV)?an imbalance of the vaginal microbiota?in enhancing susceptibility to different UTI outcomes, but experimental models have not explained the underlying mechanisms. One of the strongest risk factors for UTI is frequent and/or recent sexual activity. It is likely that vaginal bacteria are commonly introduced into the bladder in sexually active women. Indeed, several vaginal bacteria?most frequently Gardnerella vaginalis (G.v.) and Lactobacillus crispatus (L.c.)? have been isolated from urine collected directly from the bladder by needle aspiration. G.v. is found at high levels during BV. Although culture conditions are not often poised to detect it, its presence in urine has been associated with a history of recurrent UTI (rUTI). L.c. is widely regarded as a ?healthy? vaginal bacterium and is most often isolated from healthy women without lower urinary tract symptoms. Our new mouse model shows that when G.v. gains access to the urinary tract, it triggers exfoliation of the bladder epithelium. When mice harbored latent intracellular bladder reservoirs of E.c., bladder exposures to G.v. triggered E.c. egress and rUTI at a 4-times higher rate compared to exposures to vehicle alone or L.c. Importantly, G.v. exposures were cleared rapidly by the host and the organism was absent by the time E.c. rUTI took place. These data demonstrate that exposures of the bladder to vaginal bacteria can be transient, yet have a marked effect on bladder pathophysiology, and provide a potential explanation for the link between BV and UTI. In this proposal, we will pursue two aims to understand how other vaginal bacteria may promote or mitigate the negative effects of G.v. in the bladder. Aim 1 will investigate whether L.c. can prevent G.v.-induced bladder exfoliation and E.c. rUTI. This is particularly relevant because L.c. is often referred to in the recent literature as ?protective? in the bladder based on clinical associations. Our studies will address this possibility directly in an experimental system. Aim 2 will define how aspects of human vaginal microbiome composition influence bladder exfoliation in women and examine how G.v. acts within native communities to cause exfoliation and E.c. emergence in mice. Experiments in mice also will reveal whether taxonomic groups in addition to G.v. contribute to these effects in the bladder. Successful completion of these studies will uncover new paradigms in bacterial pathogenesis and provide novel insights that will inform future efforts to treat and prevent rUTI.